The matrix protein of bones and teeth play key roles in the structure and functions of these tissues. Our objective is to study the function of these macromolecules and to understand the regulation of their expression. Our first approach is to use isolated cells derived from the skeleton to study gene control at the nuclear level. Such experiments employ cDNA and genomic DNA isolation and cloning as well as extensive DNA gene mapping and sequencing. To understand the mechanisms that control RNA production matrix gene promoter DNA is analyzed using DNA transfection into cultured skeletal cells. DNase protection mobility shift assay, UV cross-linking and southwestern blotting are used to understand the nature of the DNA protein interactions that control matrix gene expression. To study matrix protein function, experiments are underway to create transgenic mice that are null (not able to make) specific matrix genes. Our approach is to replace specific genes by a targeting method that relies on homologous recombination in embryonic stem cells. In addition, in parallel, we are creating mice that have a "gain of function" (make more of a specific gene) using conventional transgenics. It is theorized that the combination of targeted "gene knockout" and "gain of function" transgenic animals will provide new incite into the role of matrix proteins in the development and aging of skeletal tissue.